Transcription and RNA processing Fall, 2015

Transcription Outline Notes RNA Polymerase Structures Subunits Template versus coding strands Polymerase movement. Promoters Prokaryotic sequences / -35 / -10 sequences Alternative promoters - governed by sigma factors Prokaryotic RNA Synthesis Transcription bubble

Termination signal ◦Stem loop ◦FMN-specific termination ◦Rho protein. Antibiotic inhibition ◦Rifampicin / Site of action - elongation blocker ◦Actinomycin D (binds DNA double helix - ) tRNA and rRNA Processing in prokaryotes ◦Ribonuclease P (generates 5' terminus of tRNAs) ◦Ribonuclease III (excises 5S, 16S, 23S rRNAs from primary transcript) ◦CCA addition to tRNAs ◦Base modifications Uridylate modifications

Eukaryotic RNA Synthesis Transcription/Translation - Prokaryotes vs. Eukaryotes RNA Polymerases Amanitin structure and source Promoter elements Eukaryotic TATA box / CAAT and GC boxes Transcription Initiation TATA-binding protein - DNA complex Transcription factor HSTF ◦Sequence recognized = 5' CNNGAANNTCCNNG 3' ◦Binding sites II

Enhancer sequences ◦No promoter activity of own ◦Act up to several thousand bp away from gene ◦Act upstream, downstream, in middle of gene, and orientation independent ◦Specific to specific cells Eukaryotic rRNA ◦Made by RNA Polymerase I as pre-rRNA ( and ) Eukaryotic tRNA ◦Made by RNA Polymerase III ◦Processing Eukaryotic mRNA ◦Made by RNA Polymerase

◦Capping 5' end of mRNAs Polyadenylation 3' end of mRNAs microRNAs ◦made by RNA Pol II or III ◦processed from larger precursors ◦roles in controlling gene expression RNA editing ◦apo B-100/apo B-48 ◦Cation channel proteins ◦Trypanosomes (insertion of uridines after transcription using guide RNAs)

Splicing ◦Sites ( ◦Splicing mechanism ◦Lariat branch point ◦Spliceosome assembly ◦Splicing catalytic center ◦snRNPs ◦Transcription and processing coupled ◦Splicing mutations and disease Thalassemia Examples ◦Alternative splicing patterns (calcitonin/CGRP - ) ◦Self splicing intron Schematic ◦Splicing pathway comparisons

Definition of: transcription, replication, and translaytion

RNA POLYMERASE

DNA POLYMERASE AND RNA POLYMERASE Differences and similarities Template Primers Starting point Directions Proof reading

SUBUNITS

TEMPLATE VERSUS CODING STRANDS

PROMOTERS

-35/-10 SEQUENCES

Alternative promoters - governed by sigma factors

Prokaryotic RNA Synthesis Transcription bubble

phases Initiation: the first 10 nt, open complex, sigma leaves. Rifampicin bind RNA pol and prevent transfer from initiation to elongation Elongation: RNA pol fits and starts Termination

Antibiotic inhibition ◦Rifampicin

Termination signal ◦Stem loop

◦FMN-specific termination

◦Rho protein

tRNA and rRNA Processing in prokaryotes

Processing One long RNA transcript, and then the individual pieces are cleaved Ribonuclease P: chewing away 5 ‘ end and to make 5’ end of tRNA Ribonuclease III: cuts the 5s, 16s, and the 23s RNA CCA sequence is added to the 3’ end of tRNA

◦Actinomycin D (binds DNA double helix

◦Base modifications Uridylate modifications

tRNA is largly modified rRNA little modified mRNA very very little modified

Eukaryotic RNA Synthesis

Transcription/Translation - Prokaryotes vs. Eukaryotes

RNA Polymerases

Amanitin structure and source

Promoter elements

Eukaryotic TATA box :any between -35 to -100, not always t but genes which are made largely they will have it

CAAT and GC boxes

Enhancer sequences – No promoter activity of own – Act up to several thousand bp away from gene – Act upstream, downstream, in middle of gene, and orientation independent – Specific to specific cells

Transcription Initiation

TATA-binding protein - DNA complex

Transcription factor HSTF ◦Sequence recognized = 5' CNNGAANNTCCNNG 3' ◦Binding sites

Eukaryotic rRNA ◦Made by RNA Polymerase I as pre-rRNA

Eukaryotic tRNA ◦Made by RNA Polymerase III ◦Processing

Eukaryotic mRNA ◦Made by RNA Polymerase II ◦Capping 5' end of mRNAs : protective against nucleases, a handle for translation, and clear 5’ end

Polyadenylation 3' end of mRNAs

microRNAs ◦made by RNA Pol II or III ◦processed from larger precursors ◦roles in controlling gene expression

RNA editing ◦apo B-100/apo B-48 ◦Cation channel proteins ◦Trypanosomes (insertion of uridines after transcription using guide RNAs)

Splicing

◦Sites

◦Splicing mechanism

◦Alternative splicing patterns (calcitonin/CGRP

◦Spliceosome assembly

◦Splicing catalytic center

◦snRNPs

◦Splicing mutations and disease Thalassemia

Examples

◦Transcription and processing coupled